In bacteria a unique modification takes place where the sulfur of a 5-methyl-aminomethyl-2-thiouridine nucleotide in tRNA Glu,Lys,Gln gets replaced with selenium (Se). This specific incorporation process is catalyzed by the enzyme tRNA 2-selenouridine synthase (tRNA 2-SUS). The known biosynthetic steps involve the synthesis of selenophosphate (which is the universal biological Se donor for the tRNA modification and for incorporation of Se into proteins) from ATP, selenide, and selenophosphate synthetase. The specific mechanism for this modification is not known, but recent work shows that thiophosphate inhibits in vitro activity 100% at 100 microM and 50% at 10 microM. Additionally, in vitro assays using 35S-labeled tRNA substrate gave a weak, but reproducible, radioactive thiophosphate peak on the HPLC profile when cold carrier thiophosphate is added after the reactions. The specific radioactivity of the tRNA's needs to be greater than what has been currently developed (1,000,000 CPM/40 microg tRNA) to accurately interpret the radiolabel experiments. These experiments suggest that the mechanism of replacement of sulfur with selenium may involve a nucleophilic exchange reaction where selenophosphate attacks the C=S moiety and thiophosphate is the product. The availability of an E. coli overproducing strain of tRNA Glu has facilitated the project. This strain has been used to incorporate 35S into the tRNA's and may be useful in the purification of tRNA 2-SUS. In vivo incorporation of 75Se into the tRNA's from the overproducing strain indicates that the cellular machinery accommodates the increased amount of substrate. It will be important to determine if the overproduction of the tRNA substrate will regulate and perhaps increase the expression of tRNA 2-SUS, and to see if this is further regulated by the presence of Se.